Drug addiction is a chronic, relapsing disorder in which drug-related associations (e.g., discrete drug cues, locations in which drugs were consumed, and drug paraphernalia) are capable of exerting tremendous control over behavior long after drug taking has ceased. A hallmark feature of drugs of abuse is that they result in persistent, long-lasting functional and structural alterations in brain reward circuits such as the nucleus accumbens. Recent discoveries have revealed that epigenetic modifications, such as methylation of cytosine nucleotides in DNA, are key regulators of long-term synaptic plasticity, learning, and long-term or even transgenerational behavioral change. Moreover, novel findings indicate that drugs of abuse such as cocaine induce epigenetic changes in the nucleus accumbens, and that these changes control cocaine-related neuroadaptations. However, very little is known about how experience with cocaine alters DNA methylation within the nucleus accumbens, and whether this modification subserves cocaine-induced neuronal and behavioral plasticity. This proposal will examine whether changes in DNA methylation (and a recently discovered brain-enriched intermediate modification, DNA hydroxymethylation) are induced by cocaine experience, and whether these changes are meaningfully related to cocaine-related behaviors. Changes in DNA methylation and hydroxymethylation will be investigated using a variety of cutting-edge techniques, including methylated DNA immunoprecipitation, direct bisulfite sequencing of DNA, and whole-genome next generation sequencing. Additional studies will examine for the first time whether these changes occur within individual cell populations in the nucleus accumbens. These assays will allow us to determine not only whether cocaine experience is associated with changes in DNA methylation in the nucleus accumbens, but will also reveal which genes and which neuronal subtypes such changes are affecting. Furthermore, the ability of DNA methylation changes to functionally modulate cocaine-induced behavioral plasticity will be examined by blocking or overexpressing critical DNA methylation and demethylation machinery during cocaine exposure. The results will provide groundbreaking insight into the epigenetic control of cocaine-related neuroadaptations and enhance our understanding of the molecular pathways that regulate motivated behavior.